Gem-diphosphonic acids, and pharmaceutical compositions containing them

ABSTRACT

Diphosphonic acids of formula (I), wherein R 1  and R 2  are hydrogen or C 1  -C 4  alkyl; (A) is hydrogen, halogen, hydroxy or C 1  -C 12  alkyl; (B) is a bond, a C 1  -C 8  alkylene chain, a cycloalkylalkylene chain, an alkylene chain substituted by cyclohexyl or cyclopentyl groups, or an aralkyl chain, an alkyl chain containing an heteroatom (O, S or N--CH 3 ) or an ureido residue --(CH 2 ) nl  --NHCONH--(CH 2 ) n  --with n ranging from 1 to 5, R 3  is hydrogen, C 1  -C 9  alkyl, C 3  -C 6  cycloalkyl, benzyl, phenyl or p-methoxybenzyl; (C) is C 1  -C 5  alkyl, phenyl or an aralkyl chain; R 4  is hudrogen, C 1  -C 5  alkyl or an amino group optionally substituted by alkyl, phenyl, benzyl, p-methoxybenzyl, acyl, amminoacidic or peptide groups; R 5  and R 6  are 2-haloethyl or together form a 1-aziridinyl residue, are useful as antitumor agent.

The present invention relates to diphosphonic acids having antitumoractivity, a process for the preparation thereof and pharmaceuticalcompositions containing them.

Gem-diphosphonic acids and the salts thereof are known and used in thetreatment of osteoporosis and bone resorption (EP 96.931, EP 252.504, BE896.453, BE 903.519, DE 3.016.289, DE 3.540.150, DE 2.534.391).Moreover, diphosphonic acid esters having pesticide activity aredisclosed in U.S. Pat. No. 3.906.062. However, no compounds described inthe above mentioned patents have been reported to have intrinsicantitumor activity.

DE 3.425.812 discloses 1,1-diphosphonic acid derivatives having abis[(haloalkyl)amino]phenyl residue as agents useful for the treatmentof bone tumours. In fact, beside having the bone tropism characteristicof diphosphonic acids, they also have the typical cytotoxic activity ofmolecules bearing dialkylating functions.

It has now been found that diphosphonic acid derivatives characterizedby the presence of a bond which can be physiologically hydrolyzed,connecting the diphosphonic derivative with a dialkylating residue,have, compared with the above cited compounds, advantageous antitumorand antimetastatic properties, which could not be predicted on the basisof their chemical structure and of the presumed bioconversion thereofinto the separate components (diphosphonic derivative and alkylatingderivative).

The present invention, therefore, provides compounds of formula (I)##STR1## wherein: R₁ and R₂, which can be the same or different, arehydrogen or C₁ -C₄ alkyl;

(A) is hydrogen, halogen (chlorine, bromine or iodine), hydroxy,straight or branched C₁ -C₁₂ alkyl;

(B) is a covalent bond, a straight or branched C₁ -C₈ alkylene or,together with the adjacent nitrogen atom, a group of formula ##STR2##therein the groups --N(R₃)-- and --(CA₂)_(n) -- may be in the 1,1; 1,2;1,3 or 1,4 position of the ring; an ortho, meta or para -substitutedaralkylene of formula ##STR3## an alkylene chain containing at least onehetero-atom of formula --[CH(CH₃)]_(p) --(CH₂)_(nl) --X--(CH₂)_(n) --; mis the integer 5 or 6; n and n₁ are an integer from 1 to 5; p is zero or1 and X is O, S, ##STR4## or the ureido group --NH--CO--NH--; R₃ ishydrogen, straight or branched C₁ -C₉ alkyl, C₃ -C₆ cycloalkyl, benzyl,phenyl or p-methoxybenzyl;

(C) is straight or branched C₁ -C₅ alkyl, phenylene, an aralkylene chainof formula ##STR5## in which n is as above defined; R₄ is hydrogen,straight or branched C₁ -C₄ alkyl, or it is a group of formula ##STR6##in which R₇ and R₈, which are the same or different, are hydrogen,straight or branched C₁ -C₆ alkyl, phenyl, benzyl, p-methoxybenzyl, orone of R₇ and R₈ is as above defined and the other one is a group offormula ##STR7## in which R₉ is hydrogen, straight or branched C₁ -C₄alkyl, phenyl, benzyl, p-methoxyphenyl, straight or branched C₁ -C₄alkoxy, halo-C₁ -C₄ -alkoxy; R₅ and R₆ are haloethyl (2-chloroethyl,2-bromoethyl, 2-iodoethyl) or R₅ and R₆, together with the nitrogen atomto which they are bound, are a 1-aziridinyl residue of formula ##STR8##

The present invention also includes racemic and diastereoisomericmixtures as well as the single enantiomers and diastereoisomers of thecompounds of formula (I).

The present invention further comprises the pharmaceutically acceptablesalts of the compounds of formula (I), for example with inorganic bases,such as the salts with alkali metals (e.g. sodium or potassium) or withalkaline-earth metals (e.g. calcium or magnesium), or the ammoniumsalts; the salts with organic bases, such as methylamine, ethylamine,propylamine, isopropylamine, butylamine, t-butylamine, dimethylamine,diethylamine, diethanolamine, trimethylamine, triethylamine, piperidine,pyridine, picoline, dicyclohexylamine; the organic acid addition salts,such as: formate, acetate, trifluoroacetate, maleate, fumarate,tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate salts;the inorganic acid addition salts, such as hydrochloride, hydrobromide,sulphate, hydrogen sulphate, phosphate salts; or the salts with aminoacids, such as aspartates, glutamates or the salts with lysine orarginine.

C₁ -C₄ Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl or isobutyl; particularly preferred are methyl and ethyl. The C₁-C₁₂ alkyl group can be, beside the meanings above precised for C₁ -C₄alkyl, n-pentyl, n-hexyl, n-decyl and the like; particularly preferredare methyl and ethyl. The alkylene chain (B) is preferably --(CH₂)_(q)--, wherein q is an integer from 2 to 5, ##STR9## wherein r is aninteger from 2 to 5, or one of the groups of formulae ##STR10## whereins is an integer from 1 to 4.

R₃ is preferably hydrogen or methyl, whilst (C) is preferably benzyl or##STR11##

When (C) is benzyl, R₄ is preferably a group of formula ##STR12##wherein R₇ and R₈ are preferably hydrogen, or one of them is R₉ --CO--wherein R₉ is hydrogen, methyl, tert-butoxy, trichloromethoxy,(2,2,2)trichloroethoxy, benzyloxy, ethoxy.

When (C) is a residue of formula ##STR13## R₄ is preferably hydrogen.

R₅ and R₆ are preferably 2-chloroethyl.

The compounds according to formula (I) are prepared by reaction of acompound of formula (II) ##STR14## wherein R₅, R₆ and (C) are as abovedefined and R'₄ is the same as R₄ or a group which can be transformedinto R₄ by removal of any protecting groups present, T is hydroxy or agroup which activates the carboxylic function with a compound of formula(III) ##STR15## wherein R₁, R₂, R₃, (A) and (B) are as above defined, togive a compound of formula (Ia) ##STR16## wherein R₁, R₂, R₃, R₄, R₅,R₆, (C), (B) and (A) are as above defined, which compound can in itsturn be transformed into a compound of formula (I) by means ofwell-known reactions for the selective elimination of the protectinggroups, or by alkylating or acylating the amino groups, and the like.

When in the reaction of compounds (III) with compounds (II) the latterare used in form of carboxylic acids (T═OH), the reaction is generallycarried out in the presence of a condensing agent such asN,N'-dicyclohexylcarbodiimide,N-cyclohexyl-N'-morpholinoethylcarbodiimide,N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide;N,N'-carbonyl-bis(imidazole); phosphorus oxychloride; phosphorustrichloride; thionyl chloride; oxalyl chloride; ethyl chloroformate;isobutyl chloroformate; morpholinoethylisonitrile and the like. Examplesof activated forms of carboxylic acids, according to formula (III), areacid halides, symmetrical or mixed anhydrides (e.g. withmethanesulfonic, acetic, isobutyric, pivalic, trichloroacetic acids);activated amides (e.g. with imidazole or triazole); acylazide; activatedesters (e.g. p-nitrophenyl ester, methoxymethyl ester, 2,4-dinitrophenylester, pentachlorophenyl ester, hydroxysuccinimido ester,1-hydroxy-2-(1H)-pyridone ester, 1-hydroxybenzotriazole ester) and thelike. The reaction can be carried out in the presence of an inorganicbase, such as an alkali carbonate or hydrogen carbonate, an alkaline oralkaline-earth hydroxide or an organic base, such as triethylamine,tributylamine, pyridine, dimethylaminopyridine, N-alkylmorpholine,N,N-dialkyl-aniline and the like.

The reaction can be carried out at a temperature ranging from -40° C. tothe reflux temperature of the solvent, preferably using a slight molarexcess of compound (II) to compound (III), in a solvent such as water,pyridine or N,N-dimethylformamide or mixtures thereof.

The reaction temperature preferably ranges from -10° C. to roomtemperature and the reaction time ranges from 1 to 48 hours, butgenerally the reaction is complete within 2-12 hours.

Alternatively, the mixed anhydrides or the acid chlorides of thecompounds of formula (II) can be reacted with the compounds of formula(III) in heterogeneous phase, in a solvent such as an ester, e.g. methylor ethyl formates; ethyl, methyl or isopropyl acetates; or in ahalogenated solvent such as dichloromethane or chloroform, or in astraight or cyclic ether such as dioxane, tetrahydrofuran, ethyl ether,tert-butylmethyl ether and mixtures thereof. Temperature conditions andreaction times are the same as above reported.

Compounds of general formula (II) are known compounds, which arecommercially available and/or can be prepared by means of conventionalmethods, such as those described in: J. Med. Chem. 24, 1304, (1981); CA51: 8066d, (1957); BE 905,974; CA 104: 141897 (1986); J. Med. Chem. 7,468, (1964); J. Med. Chem. 6, 85, (1963); Cancer Chem. Rep. 50, 685,(1966); J. Med. Chem. 21, 16, (1977); J. Org. Chem. 26, 1554, (1961); J.Org. Chem. 26, 1674, (1961); CA 64: 10267g, (1966); J. Chem. Soc., 2994,(1960); Biochem. Pharmacol. 11, 847, (1962); Biochem. Pharmacol. 12,833, (1963); CA 73: 131293c, (1970); Biochem. Pharmacol. 5, 192, (1960).

Compounds of general formula (III) are also known and/or can be preparedaccording to known methods. See, for instance, EP 96,931, EP 252,504, BE903,519, DE 3,016,289, EP 224,751, DE 2,534,391, EP 197,478.

If desired, compounds of general formula (I) wherein R₁ and R₂ aredifferent from hydrogen, can optionally be transformed into thecorresponding gem-diphosphonic acids by treatment with a molar excess oftrialkylsilyl-chloride, -iodide or -bromide in a halogenated solventsuch as dichloromethane, 1,2-dichloroethane, 1,1,2-trichloroethane andthe like. Trimethylsilyl iodide is preferably used.

The reaction times ranges from a few minutes to 72 hours; the reactiontemperatures range from 0° C. to the solvent's reflux temperature;preferred reaction conditions are those according to J. Org. Chem: 28,2975-78, (1963).

The removal of the secondary- or primary-amine protecting groupsoptionally present in the compounds of general formula (I) can becarried out according to well-known techniques, particularly those usedin peptide synthesis.

The compounds of the invention have high cytotoxic activity againsttumour cells, as it can be evidenced by means of "in vitro" testscarried out, for instance, according to the procedure described by M. P.Hacker, Cancer Res. 45, 4748, (1985). The ID₅₀ (i.e. the compound dosewhich can inhibit by 50% the growth of "in vitro" cultured murine andhuman tumour cells of both solid and hematic tumors) of the compounds ofthe invention were found to be comprised from 0.1 to 5 μg/ml of culturemedium. Under these test conditions, the compounds of the invention:N-{4-[bis(2-chloroethyl)amino]phenyl-(L)-alanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid monohydrochloride andN-{4-[bis(2-chloroethyl)amino]phenyl-butirroyl}-4-amino-1-hydroxy-butane-1,1-diphosphonicacid have an ID₅₀ of 0.1 μg/ml and 0.5 μg/ml, respectively, againstmurine leukemia L1210.

"In vitro" studies on the compound of the inventionN-{4-[bis(2-chloroethyl)amino]phenyl-(L)-alanyl}-4-amino-1-hydroxy-butane-1,1-diphosphonic,using different human tumor cell lines (for example fibrosarcoma HT1080, osteosarcoma H2-OS etc.) showed ID₅₀ ranging from 13.6 to 16.5μg/ml; the substance appears to be less cytotoxic than the relatedstarting material 4-amino-1-hydroxy-1,1-diphosphonic acid, whose ID₅₀range from 2.7 to 8.2 μ/ml.

The same compound, when tested "in vivo" by i.v. administration (on days2--6--9--13 after tumor implantation) in rats bearing Walker B mammarycarcinoma (implanted on day 0 intramuscolarly 10⁶ cells) causes completeregression of tumor growth combined with normalization of tumor inducedhypercalcemia. Under the same experimental conditions, the relatedstarting diphosphonate (i.e. 4-amino-1-hydroxy-butane-1,1-diphosphonicacid) even if endowed with higher "in vitro" cytotoxicicy, issurprisingly ineffective in retarding tumor growth, also whenadministered at its maximum tolerated dosage.

The compounds of the invention are characterized by a low acute toxicityand are well tolerated by the animals.

The compounds of the invention have a high therapeutical index, in lightof the low toxicity and the effective antitumor activity thereof.Moreover, the high water-solubility of the compounds of the presentinvention allows the easy preparation of parenteral and oralpharmaceutical forms.

The compounds of formula (I), when administered to humans and animalsaffected with tumours which can be treated with alkylating agents, atdoses ranging from 1 mg to 1.2 g/m² body area, can induce the regressionof the above mentioned tumoral forms.

The effective dosage for the compounds of the invention can bedetermined by the expert clinicians according to conventional methods.The relationship between the dosages used for various animal species andthose for humans (on the basis of mg/m² body area) is described byFreireich, E. J., et al., "Quantitative Comparison of Toxicity ofAnticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man", CancerChemother. Rep., 50, n. 4, 219-244, May 1966.

Nevertheless, the patient will be administered generally with doses from1 to 1,200 mg/kg body weight of the compounds of the invention, with adosage regimen which will vary depending on various factors, which arewell known to the expert clinicians.

Some compounds of the invention may have such an high toxicity, or suchan unfavourable therapeutical index, so as to be unsuited for anantitumor treatment in the patients. Nevertheless, those parameters caneasily be determined by means of conventional toxycological tests, suchas acute and subacute DL₅₀ in mouse. Of course, those compounds whichturn out to be toxic will be avoided.

The compounds of the invention will be used in the treatment of thosetumours which can be treated with alkylating agents.

Particularly, multiple myeloma, osteosarcoma; bone-metastasis; breast,ovarian and testis carcinomas can also be advantageously treated.

Moreover, the compounds of the invention can advantageously be used inthe therapy of other solid and hematic neoplasias, such as lymphomas andleukemias in humans and animals, according to treatment protocols whichcan be easily determined by those skilled in the art.

The compounds of the invention are preferably administered by theintravenous or intraarterial routes, even though other administrationforms can be envisaged in particular cases.

The pharmaceutical forms which can be used for parenteral administrationinclude sterile aqueous solutions or sterile powders for the extemporarypreparation of solutions, as well as oily preparations for intramuscularor intraperitoneal administrations.

Other useful pharmaceutical forms are syrups or similar liquid forms, aswell as solid forms such as tablets, capsules and the like.

The following examples illustrate the invention in more detail.

EXAMPLE 1

a) A solution of bis(tert-butoxy)carbonate (450 mg) in tetrahydrofuran(THF; 4 ml) is quickly added to a solution of4-[bis(2-chloroethyl)amino]-(L)-phenylalanine (300 mg) in THF (10 ml)and 1N NaOH (1 ml).

The resulting mixture is stirred at room temperature for 2 hours,adjusting pH to about 9 by repeated additions of 1N NaOH. Then thesolvent is removed under vacuum and the residue is partitioned betweenwater (3 ml) and ethyl ether (5 ml).

The organic phase is discharged and the aqueous one is acidified withHCl and repeatedly extracted with ethyl ether. The combined organicextracts are dried over Na₂ SO₄ and evaporated to dryness under vacuum,to obtain a foamy residue ofN'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)phenylalanine(290 mg).

NMR (CDCl₃, TMS) δ=1.4 (s, 9H); 3.1 (m, 2H); 3.65 (m 8H); 4.55 (m, 1H);4.99 (d, 1H); 6.62 (d, 2H); 7.12 (d, 2H); 8.1 (s, 1H).

b) N-Hydroxysuccinimide (173 mg) and morpholinoethylisonitrile (0.133ml) are added to a solution of the compound from step a) (290 mg). Themixture is stirred at room temperature for one hour, then concentratedto dryness under vacuum. The residue is taken up into 2N HCl (5 ml) andrepeatedly extracted with ethyl ether (3×5 ml). The organic phases arecollected, washed with 5% aqueous NaHCO₃ ; with water (5 ml) and thendried over Na₂ SO₄. Upon removing the solvent under vacuum, an oilyresidue is obtained which is crystallized from an ethyl ester--isopropylether mixture to give 260 mg ofN'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalaninehydroxysuccinimido ester, melting point 140°-143° C.;

NMR (TMS, CDCl₃) δ=1.45 (s, 9H); 2.85 (s, 4H); 3.15 (m, 2H); 3.69 (m,8H); 4.9 (m, 1H); 6.61 (d, 2H); 7,15 (d, 2H).

c) A solution of the obtained activated ester (50 mg) indimethylformamide (DMF, 2.5 ml) is slowly dropped into a solution of5-amino-1-hydroxypentane-1,1-diphosphonic acid (21.8 mg) dissolved in amixture of water (2.5 ml), 1N NaOH (0.331 ml) and DMF (2 ml). DMF isadded to the reaction mixture simultaneously to the ester solution,until complete dissolution of the reagents is obtained (2.5 ml).

The obtained slightly opalescent solution is centrifuged; thesupernatant liquid is collected and concentrated under reduced pressure.The residue is diluted with DMF (5 ml) to separate a precipitate whichis collected by centrifugation and washed with ethyl acetate, yielding35 mg ofN-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt, m.p. >260° C.

NMR (TMS, D₂ O): δ1.3 (s, 9H); 1.4-1.7 (m, 4H); 1.8-2.05 (m, 2H);2.62-3.1 (m, 2H); 3.2 (t, 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H);7.15 (d, 2H).

EXAMPLE 2

Following the same procedure as described in Example 1, by reactingN'-(tertbutoxyarbonyl)-4-8 bis-(2-chloroethyl)amino]-(L)-phenylalaninehydroxysuccinimido ester with the appropriateaminoalkyl-1-hydroxy-1,1-diphosphonic acids, the following compounds areobtained:

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.6 (m, 2H), 1.92 (m, 2H); 2.62÷3.1 (m,2H); 3.2 (t, 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-amino-1-hydroxypropane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.32 (s, 9H); 2.1 (m, 2H), 2.65÷3.1 (m, 2H); 3.21 (m,2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-6-amino-1-hydroxyhexane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.5 (m, 2H), 1.6 (m, 4H); 1.9 (m, 2H);2.65÷3.1 (m, 2H); 3.20 (t, 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.8 (d, 2H);7.15 (d, 2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino)]-(L)-phenylalanyl}-4-amino-1-hydroxypentane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.32 (s, 9H); 1.4 (d, 3H), 1.7 (m, 2H); 1.9 (m, 2H);2.6÷3.05 (m, 2H); 3.15 (d, 1H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H);7.15 (d, 2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(2-aminocyclopent-1-yl)-1-hydroxypropane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.42÷1.75 (m, 9H), 1.9 (m, 2H); 2.62÷3.1(m, 2H); 3.3 (m, 1H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H); 7.15 (d,2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-pentamethylene-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.55 (m, 10H), 1.6 (m, 2H); 1.9 (m, 2H);2.62÷3.1 (m. 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H);

N-methyl-N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.55 (m, 4H), 1.9 (m, 2H); 2.1 (m, 3H);2.62÷3.1 (m, 2H); 3.2 (t, 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H);7.15 (d, 2H);

N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-tetramethylene-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (D₂ O, TMS): δ=1.3 (s, 9H); 1.5 (m, 8H), 1.6 (m, 2H); 1.9 (m, 2H);2.62÷3.1 (m, 2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H);

EXAMPLE 3

Following the same procedure as described in Example 1b), by reacting4-[4-[bis(2-chloroethyl)amino]phenyl]butyric acid (300 mg) withN-hydroxysuccinimide (245 mg) and N-morpholinoethylisonitrile (0.18 ml),4-[4-[bis(2-chloroethyl)amino]phenyl]butyric acid N-hydroxysuccinimidoester (360 mg) is prepared, m.p. 80°-82° C.

Following the same procedure as described in Example 1c), the abovehydroxysuccinimido ester (112 mg) is reacted with5-amino-1-hydroxypentane-1,1-diphosphonic acid (61.2 mg) to obtainN-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt (80 mg), m.p. >260° C.

NMR (TMS, D₂ O): δ=1.4-1.7 (m, 4H); 1.8÷2.05 (m, 4H), 2.15 (t, 2H); 2.55(t, 2H); 3.18 (t, 2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

HPLC: Partisfere® C₁₈, 150×4.6 mm; 0.025M sodium heptanesulfonate inwater/acetonitrile/dioxane 70:20:10, pH ˜2.5 with H₃ PO₄ ; flow: 1.3ml/min; λ=255 nm. Retention time: 6.86'.

EXAMPLE 4

Following the procedure described in Example 3, by reactingN-4-[4-[bis(2-chloroethyl)amino]phenyl]butyric acid hydroxysuccinimidoester with the appropriate aminoalkyl-1-hydroxy-1,1-diphosphonic acids,the following compounds are obtained:

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.7÷2.05 (m, 6H); 2.25 (t, 2H); 2.55 (t, 2H); 3.18(t, 2H); 3.75 (s, 8H); 6.85 (d, 2H); 7.2 (d, 2H);

HPLC: Partisfere® C₁₈, 150×4.6 mm; 0.025M sodium heptanesulfonate inwater/acetonitrile/dioxane 70:20:10, pH ˜2.5 with H₃ PO₄ ; flow 1.3ml/min.; λ=255 nm. Retention time: 5.96'.

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-3-amino-1-hydroxypropane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.8÷2.05 (m, 4H); 2.25 (t, 2H); 2.55 (t, 2H); 3.18(t, 2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-6-amino-1-hydroxyhexane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.4 (m, 2H); 1.6 (m, 4H); 1.9 (m, 4H); 2.25 (t, 2H);2.55 (t, 2H); 3.18 (t, 2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-amino-1-hydroxypentane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.4 (d, 3H); 1.7 (m, 2H); 1.9 (m, 4H); 2.25 (t, 2H);2.55 (t, 2H); 3.18 (d, 1H); 3.75 (s, 8H); 6.85 (d, 2H); 7.2 (d, 2H);

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-3-(2-aminocyclopent-1-yl)-1-hydroxypropane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.42÷1.75 (m, 9H); 1.9 (m, 4H); 2.25 (t, 2H); 2.55(t, 2H); 3.2 (m, 1H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-amino-4,4-pentamethylene-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.55 (m, 10H); 1.6 (m, 2H); 1.9 (m, 4H); 2.15 (t,2H); 2.55 (t, 2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

N-methyl-N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.55 (m, 4H); 1.9 (m, 4H); 2.1 (s, 3H); 2.15 (t, 2H);2.55 (t, 2H); 3.18 (t, 2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-amino-4,4-tetramethylene-1-hydroxybutane-1,1-diphosphonicacid (trisodium salt),

NMR (TMS, D₂ O): δ=1.5 (m, 8H); 1.7÷2.05 (m, 6H); 2.15 (t, 2H); 2.55 (t,2H); 3.75 (s, 8H); 6.8 (d, 2H); 7.15 (d, 2H);

EXAMPLE 5

A solution ofN-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt (35 mg) in methanol saturated with hydrochloric acid(2 ml) is heated to 40°-50° C. for 4 hours, then the solution isconcentrated to reduced volume and the precipitated sodium chloride isfiltered. The filtrate is evaporated to dryness and the residue istriturated with acetone and filtered. After recrystallization fromethanol/ethyl ether,N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid monohydrochloride (25 mg) is obtained, m.p. >260° C., [α]_(D) =+7.9(c=2, methanol), NMR (TMS, D₂ O): 1.25÷1.40 (m, 2H), 1.42÷1.72 (m, 2H);1.8÷2.06 (m, 2H); 2.9÷3.2 (m, 4H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (t,1H); 7.35 (s, 4H).

HPLC: Partisfere® C₁₈, 150×4.6 mm; 0.025M sodium heptanesulfonate inwater/acetonitrile/dioxane 70:20:10, pH ˜2.5 with H₃ PO₄ ; flow 1.3ml/min.; λ= 255 nm. Retention time: 4.61'.

EXAMPLE 6

Following the same procedure as described in Example 5, by reacting thetrisodium salts of the acids described in Example 2, the following acidmonohydrochloride salts are obtained:

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.6 (m, 2H); 1.92 (m, 2H); 3.15 (m, 4H); 3.65(t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-amino-1-hydroxypropane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=2,1 (m, 2H); 3,15 (m, 4H); 3,65 (t, 4H); 4,0(t, 4H); 4,1 (m, 1H); 7,35 (s, 4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-6-amino-1-hydroxyhexane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.5 (m, 2H); 1.6 (m, 4H); 1.9 (m, 2H); 3.15 (m,4H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxypentane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.4 (m, 3H); 1.7 (m, 2H); 1.9 (m, 2H); 3.15 (m,3H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(2-aminocyclopent-1-yl)-1-hydroxypropane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.5 (m, 8H); 1.6 (m, 2H); 1.9 (m, 2H); 3.15 (t,2H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-pentamethylene-1-hydroxybutane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.45 (m, 10H); 1.6 (m, 2H); 1.9 (m, 2H); 3.15(t, 2H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H);

N-methyl-N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.55 (m, 4H); 1.9 (m, 2H); 2.1 (s, 3H); 3.05(t, 2H); 3.15 (t, 2H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s,4H);

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-tetramethylene-1-hydroxybutane-1,1-diphosphonicacid: NMR (D₂ O, TMS): δ=1.5 (m, 8H); 1.6 (m, 2H); 1.9 (m, 2H); 3.15 (t,2H); 3.65 (t, 4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H).

EXAMPLE 7

A solution of 4-[bis(2-chloroethyl)amino]-(L)-phenylalanine (150 mg) informic acid (1.82 ml) and acetic anhydride (0.64 ml) is stirred at roomtemperature for 3 hours. The reaction mixture is concentrated to reducedvolume under reduced pressure and partitioned between water (3 ml) andethyl acetate (2×50 ml). The organic phase is dried over sodium sulphateand the solvent is evaporated under vacuum, to giveN-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanine (160 mg) as ayellow foam, [α]_(D) =+67° (C=2, ethanol).

NMR (CDCl₃, TMS): =2.9÷3.1 (m, 2H); 3.6 (m, 8H); 4.68÷4.79 (m, 1H); 6.5(m, 2H+1H); 6.69 (d, 2H); 8.1 (s, 1H).

Following the same procedure as described in Example 1b) and 1c), thiscompound is transformed into the hydroxysuccinimido ester (50 mg) andthen is reacted with 4-amino-1-hydroxybutane-1,1-diphosphonic acid (24.1mg), to obtainN-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid trisodium salt (32 mg); [α]_(D) =+7.5° (c=2, water)

NMR (D₂ O, TMS): δ=1.7÷2.0 (m, 4H); 2.9÷3.1 (m, 2H); 3.2 (m, 2H); 3.75(s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05 (s, 1H).

HPLC: Partisfere® C₁₈, 150×4.6 mm; 0.025M sodium heptanesulfonate inwater/acetonitrile/dioxane 70:20:10, pH ˜2.5 with H₃ PO₄ ; flow 1.3ml/min.; λ=255 nm. Retention time: 2.9'.

EXAMPLE 8

Following the same procedure as described in Example 1c), by reactingN-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalaninehydroxysuccinimido ester with the appropriateaminoalkyl-1-hydroxy-1,1-diphosphonic acids, the following compounds areobtained:

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.4÷1.6 (m, 4H); 1.92 (m, 2H); 2.9÷3.1 (m, 2H); 3.2(m, 2H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05(s, 1H);

N-{N'-formyl-[4-bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-amino-1-hydroxypropane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=2,1 (m, 2H); 2,9÷3,1 (m, 2H); 3,2 (m, 2H); 3,75 (s,8H); 4,55 (t, 1H); 6,85 (d, 2H); 7,15 (d, 2H); 8,05 (s, 1H);

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-6-amino-1-hydroxyhexane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.5 (m, 2H); 1.9 (m, 2H); 2.9÷3.1 (m, 2H); 3.2 (m,2H); 3.75 (s, 8H); 4.2 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05 (s,1H);

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.4 (d, 3H); 1.7 (m, 2H); 1.9 (m, 2H); 2.9÷3.1 (m,2H); 3.2 (m, 2H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d,2H); 8.05 (s, 1H);

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(2-aminocyclopent-1-yl)-1-hydroxypropane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.42÷1.75 (m, 9H): 1.9 (m, 2H); 2.9÷3.1 (m, 2H); 3.3(m, 1H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05(s, 1H);

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-tetramethylene-1-hydroxybutane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.5 (m, 8H); 1.6 (m, 2H); 1.9 (m, 2H); 2.9÷3.1 (m,2H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05 (s,1H);

N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-pentamethylene-1-hydroxybutane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.45 (m, 10H); 1.6 (m, 2H); 1.9 (m, 2H); 2.9÷3.1 (m,2H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d, 2H); 8.05 (s,1H);

N-methyl-N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt;

NMR (D₂ O, TMS): δ=1.55 (m, 4H); 1.9 (m, 2H); 2.1 (s, 3H); 2.9÷3.1 (m,2H); 3.2 (m, 2H); 3.75 (s, 8H); 4.55 (t, 1H); 6.85 (d, 2H); 7.15 (d,2H); 8.05 (s, 1H);

EXAMPLE 9

A solution ofN-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)phenylalanyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid trisodium salt (32 mg) in methanol saturated with hydrochloric acid(1 ml) is heated to 40°-50° C. for 4 hours, then the solution isconcentrated to reduced volume and the precipitated sodium chloride isfiltered. The filtrate is evaporated to dryness and the residue istriturated with acetone and filtered. After recrystallization fromethanol/ethyl ether,N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-1-hydroxybutane-1,1-diphosphonicacid monohydrochloride (25 mg) is obtained, m.p. >260° C.; [α]_(D) =+7.8(c=2, methanol).

NMR (D₂ O, TMS): δ=1.6 (m, 2H); 1.92 (m, 2H); 3.15 (m, 4H); 3.65 (t,4H); 4.0 (t, 4H); 4.1 (m, 1H); 7.35 (s, 4H).

HPLC: Partisfere® C₁₈, 150×4.6 mm; 0.025M sodium heptanesulfonate inwater/acetonitrile/dioxane 70:20:10, pH ˜2.5 with H₃ PO₄ ; flow 1.3ml/min.; λ=255 nm. Retention time: 4.24'.

EXAMPLE 10

A solution of 4-[4-[bis(2-chloroethyl)amino]phenyl]butyric acidN-hydroxysuccinimido ester (73 mg) in DMF (1 ml) is dropped at roomtemperature into a stirred solution of 4-aminobutane-1,1-diphosphonicacid tetraethyl ester (52 mg) in DMF/water 5/1 (0.6 ml).

After 3 hours, the mixture is concentrated under reduced pressure andpartitioned between a 5% sodium hydrogen carbonate solution (5 ml) andethyl acetate (5 ml). After separation of the organic phase, the aqueousphase is further extracted with ethyl acetate (2×5 ml) and discarded;the combined organic extracts are dried over sodium sulphate and solventis evaporated off under reduced pressure, to yieldN-[4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl]-4-aminobutane-1,1-diphosphonicacid tetraethyl ester (90 mg) as a clear oil;

NMR (CDCl₃, TMS): δ=1.35 (t, 12H); 1.90 (t, 2H); 2.2 (t, 2H); 2.0÷2.5(m, 5H); 2.55 (t, 2H); 3.45 (q, 2H); 3.65 (m, 8H); 4.20 (m, 8H); 6.51(t, 1H); 6.61 (d, 2H); 7.1 (d, 2H).

EXAMPLE 11

Under a nitrogen atmosphere, a solution of iodotrimethylsilane (92 μl)in anhydrous dichloromethane (1 ml) is dropped at 0° C. into a solutionofN-{4-[4-[bis(2-chloroethyl)amino]phenylbutyroyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester (72 mg) in anhydrous dichloromethane (1 ml). Themixture is stirred for 2 hours at 0° C., then it is allowed to roomtemperature and subsequently treated with methanol (1 ml). After 15minutes, the solvent is evaporated off under reduced pressure and theresidue is dissolved in water (2 ml), treated with 1N NaOH (0.228 ml)and extracted with ethyl acetate (2×2 ml). The organic extracts arediscarded. The aqueous phase is diluted with DMF to yield a white solidwhich is recovered by centrifugation, to giveN-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-aminobutane-1,1-diphosphonicacid disodium salt (45 mg);

NMR (D₂ O, TMS): δ=1.9 (m, 6H); 2.25 (t, 2H); 2.55 (t, 2H); 3.38 (m,2H); 3.72 (s, 8H); 6.85 (d, 2H); 7.2 (d, 2H).

EXAMPLE 12

Following the same procedure as described in Example 10,N-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanine-N-hydroxysuccinimidoester (87,5 mg) is reacted with 4-aminobutane-1,1-diphosphonic acidtetraethyl ester (50 mg) to obtainN-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester (87 mg) as a clear oil.

NMR (CDCl₃, TMS): δ=1.38 (m, 21H); 2.2 (m, 5H); 3.45 (m, 4H); 3.65 (m,8H); 4.18 (m, 8H); 5.05 (m, 1H); 6.51 (t, 1H); 6.61 (d, 2H); 7.1 (d,2H).

EXAMPLE 13

A solution ofN-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester (59 mg) in 10% HCl (1 ml) is heated to 45° C. forone hour, then evaporated to dryness under reduced pressure. The residueis suspended in ethyl ether (2 ml) and recovered by centrifugation, togiveN-{[4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester dihydrochloride (45 mg);

NMR (TMS, D₂ O): δ=1.22 (t, 12H); 1.95 (m, 4H); 3.2 (m, 2H); 3.4 (m,2H); 3.7 (t, 4H); 3.95 (m, 12H); 4.1 (t, 1H); 7.38 (s, 4H).

EXAMPLE 14

Under a nitrogen atmosphere, a solution of iodotrimethylsilane (134 μl)in anhydrous dichloromethane (1 ml) is dropped into a stirred solutionofN-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester (93 mg) in anhydrous dichloromethane (1 ml),cooled to 0° C.

The mixture is stirred for 2 hours, allowing it to warm to roomtemperature, then it is treated with methanol (1 ml). After 15 minutes,solvent is evaporated off under reduced pressure, the residue isdissolved in water, treated with 1N NaOH (0.254 ml) and extracted withethyl acetate (2×3 ml). The organic extracts are discarded. The aqueousphase is diluted with DMF to precipitate a solid, which is recovered bycentrifugation and washed with ethyl acetate.

45 mg ofN-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminobutane-1,1-diphosphonicacid disodium salt are obtained,

NMR (D₂ O, TMS): δ=1.95 (m, 4H); 2.9 (m, 2H); 3.4 (m, 2H); 3.65 (m, 1H);3.75 (s, 8H); 6.88 (d, 2H); 7.18 (d, 2H).

EXAMPLE 15

Following the same procedures as described in Examples 12 and 13, usingN-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenylalaninehydroxysuccinimido ester and the appropriate tetraalkylesters ofaminoalkyl-1,1-diphosphonic acids the following compounds are prepared:

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-aminopropane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-aminopentane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-6-aminohexane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-aminopentane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(2-aminocyclopent-1-yl)propane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-tetramethylene-butane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-amino-4,4-pentamethylene-butane-1,1-diphosphonicacid tetraethylester dihydrochloride;

N-methyl-N-4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-aminopentane-1,1-diphosphonicacid tetraethylester dihydrochloride;

EXAMPLE 16

Following the same procedures as described in Example 1a) and 1b),3-[bis(2-chloroethyl)amino]-(L)-phenylalanine (J. Med. Chem., 6, 85,(1963)) is transformed intoN'-(tert-butoxycarbonyl)-3-[bis(2-chloroethyl)amino]-(L)-phenylalaninehydroxysuccinimido ester, which is reacted with5-amino-1-hydroxypentane-1,1-diphosphonic acid following the proceduredescribed in Example 1c), to giveN-{N'-(tert-butoxycarbonyl)-3-[bis(2-chloroethyl)amino)-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid trisodium salt.

This compound is subsequently transformed intoN-{3-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino-1-hydroxypentane-1,1-diphosphonicacid dihydrochloride, following the procedure described in Example 5.

EXAMPLE 17

Following the procedure described in Example 1a) and 1b), N⁶,N⁶-bis(2-chloroethyl)-(DL)-lysine (J. Med. Chem. 7, 468, (1964)) istransformed into N² -(tert-butoxycarbonyl)-N⁶,N⁶-bis(2-chloroethyl)-(DL)-lysine hydroxysuccinimido ester which isreacted with 4-amino-1-hydroxybutane-1,1-diphosphonic acid, followingthe procedure described in Example 1c), to give N-[N²-(tertbutoxycarbonyl)-N⁶,N⁶-bis(2-chloroethyl)-(DL)-lysinyl]-4-amino-1-hydroxybutane-1,1-diphosphonicacid trisodium salt, which is in its turn transformed into N-[N⁶,N⁶-bis(2-chloroethyl)-(DL)-lysinyl]-4-amino-1-hydroxybutane-1,1-diphosphonicacid dihydrochloride, following the procedure described in Example 5.

EXAMPLE 18

Following the procedure described in Example 1b), by reacting4-[2-[bis(2-chloroethyl)amino]phenyl]butyric acid (J. Org. Chem. 26,1554, (1961)) with N-hydroxysuccinimide and N-morpholinoethylisonitrile,4-[2-[bis(2-chloroethyl)amino]phenyl]butyric acid N-hydroxysuccinimidoester is prepared. Said N-hydroxysuccinimido ester is reacted with4-aminobutane-1,1-diphosphonic acid tetraethyl ester, according to theprocedure described in Example 10, to giveN-{4-[2-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-aminobutane-1,1-diphosphonicacid tetraethyl ester, which is reacted with iodotrimethylsilane,following the procedure described in Example 11, to obtainN-{4-[2-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4-aminobutane-1,1-diphosphonicacid disodium salt.

We claim:
 1. Compounds of formula (I) ##STR17## wherein: R₁ and R₂,which can be the same or different, are hydrogen or C₁ -C₄ alkyl:(A) ishydrogen or hydroxy, (B) is a straight or branched C₁ -C₈ alkylene or,together with the adjacent nitrogen atom, a group of formula ##STR18##wherein the groups N(R₃) and (CH₂)_(n) may be in the 1,1; 1,2; 1,3; or1,4 position of the ring; m is the integer 5 or 6; n is an integer 1 to5; R₃ is hydrogen, straight or branched C₁ -C₉ alkyl; (C) is straight orbranched C₁ -C₅ alkylene, phenylene, an aralkylene chain of formula##STR19## in which n is as above defined; R₄ is hydrogen or a group offormula ##STR20## in which R₇ and R₈, which are the same or different,are hydrogen, straight or branched C₁ -C₆ alkyl, or one of R₇ and R₈ isas above defined and the other one is a group of formula ##STR21## inwhich R₉ is hydrogen, straight or branched C₁ -C₄ alkoxy; R₅ and R₆ are2-haloethyl or R₅ and R₆, together with the nitrogen atom to which theyare bound, are a 1-aziridinyl residue of formula ##STR22## and isomers,diastereoisomers, and pharmaceutically acceptable salts thereof. 2.Compounds as claimed in claim 1, wherein A is hydroxy.
 3. Compounds asclaimed in claim 1, wherein A is hydrogen.
 4. Compounds as claimed inclaim 1, wherein R₁ and R₂ are hydrogen.
 5. Compounds as claimed inclaim 1, wherein R₁ and R₂ are C₁ -C₄ alkyl.
 6. Compounds as claimed inclaim 1, wherein (B) is a C₂ -C₅ alkylene chain and R₃ is hydrogen. 7.Compounds as claimed in claim 1, wherein (B) is a chain of formula##STR23## wherein s is from 1 to
 4. 8. Compounds as claimed in claim 1,wherein (C) is phenylene or benzyl and R₅ and R₆ are 2-chloroethyl. 9.Compounds as claimed in claim 8, wherein (C) is benzyl and R₄ is amino,t-butoxycarbonylamino, formylamino, acetylamino, benzyloxycarbonylaminoor ethoxycarbonylamino.
 10. Compounds as claimed in claim 8, wherein (C)is phenthylene and R₄ is hydrogen.
 11. Pharmaceutical compositionsuitable for use in tumor therapy comprising at least one compound ofclaim 1 and a nontoxic carrier.